Introduction
Fecampiida, a group of parasitic platyhelminths, comprises four families (Laumer and Giribet Reference Laumer and Giribet2017; Tyler et al. 2006–Reference Tyler, Artois, Schilling, Hooge and Bush2024). Notenteridae contains two species, the polychaete-parasitic Notentera ivanovi and the octopus-parasitic Octopoxenus antarcticus (Gordeev et al. Reference Gordeev, Biserova, Zhukova and Ekimova2022; Joffe et al. Reference Joffe, Selivanova and Kornakova1997; Raikova et al. Reference Raikova, Kotikova and Frolova2017). Piscinquilinidae contains one fish-parasitic species, Ichthyophaga subcutanea (Syromjatnikova Reference Syromjatnikova1949). Urastomidae contains Urastoma cyprinae, parasitic on various bivalve species (Robledo et al. Reference Robledo, Cáceres-Martínez and Figueras1994). Fecampiidae is the most species-rich fecampiidan family, with four Fecampia, one Glanduloderma, and five Kronborgia species. Previously reported hosts for the family include myzostomids (Annelida), amphipods, barnacles, decapods, and isopods (Crustacea, Arthropoda); Sudo et al. (2021) suggested that helminth parasites found from sea slugs (Mollusca) may belong to this family (Supplementary Table S1; Kakui Reference Kakui2024b). In addition to the 14 named species, there are possibly more than 10 undescribed species in Fecampiida (e.g., Christensen Reference Christensen1981a, Reference Christensenb, Reference Christensen1988; Fiege et al. Reference Fiege, Zibrowius and Arnaud2007; Handl and Bouchet Reference Handl and Bouchet2007; Sudo et al. Reference Sudo, Hirano and Hirano2011).
Tanaidacea is a group of small aquatic crustaceans, with about 1500 species (Anderson Reference Anderson2023). Most species are free living and inhabit benthic marine habitats. Tanaidaceans are widely distributed geographically (all oceans) and vertically (intertidally to about 9000 m deep), and occasionally occur at very high densities (e.g., 140,000 individuals/m2 in Allotanais hirsutus; Delille et al. Reference Delille, Guidi, Soyer, Siegfried, Condy and Laws1985). Given their broad distribution and high abundance, tanaidaceans might serve as hosts for many parasitic organisms in aquatic ecosystems, though they have received little attention in this context, with only a few works on tanaidacean parasites published in the past decade (Błażewicz et al. Reference Błażewicz, Stępień, Jakiel, Palero, Saeedi and Brandt2020; Boyko et al. Reference Boyko, Williams and Rhodes2021; Chatterjee et al. Reference Chatterjee, Sautya, Dovgal and Gaikwad2022; Chim and Bird Reference Chim and Bird2021; Cortés et al. Reference Cortés, Campos and Bolaño-Lara2021; Jakiel et al. Reference Jakiel, Palero and Błażewicz2019; Kakui Reference Kakui2014, Reference Kakui2016; Kakui and Fujita Reference Kakui and Fujita2024; Kakui and Shimada Reference Kakui and Shimada2022).
In 2024, a tanaidacean containing vermiform organisms inside was collected around Japan. A 28S ribosomal RNA (28S) sequence determined from the parasite revealed that it is a fecampiidan flatworm, a group not previously been reported from tanaidaceans. Here, we describe the gross morphology of the fecampiidan and infer its phylogenetic position in Fecampiida based on 28S data.
Material and methods
The host tanaidacean was found in a mud sample collected with a suction sampler (slurp gun) from the deep submergence research vehicle Shinkai 6500 (Japan Agency for Marine-Earth Science and Technology; JAMSTEC) at Station 1 (Shima Spur, 33°57.0212′N, 136°53.8682′E, 794 m depth), Dive 6K#1782, on 12 June 2024 during cruise YK24-09S of RV Yokosuka (JAMSTEC). The fresh tanaidacean was photographed and then fixed and preserved in 99% ethanol. Measurements were made from digital images of the fresh individual. Body length (BL) of the host was measured from the base of the antennules to the tip of the pleotelson, and body width (BW) at the widest portion of the cephalothorax. The BL of the parasites was measured from the anterior to posterior ends, and BW at the widest part. Part of one parasite was removed from the host with sharpened needles for DNA extraction. The remaining part was deposited as a voucher specimen in the Invertebrate Collection of the Hokkaido University Museum (ICHUM), Sapporo, Japan, under catalog number ICHUM8960. DNA sequences (28S) were also determined for two cocoons of Fecampia cf. abyssicola, collected in the Kumano Sea (Stn D4: 33°59.7′N 136°56.9′E to 33°59.9′N 136°57.2′E; 802–812 m depth) on 27 June 2023 during cruise 2312 of the TRV Seisui-maru (Mie University).
DNA was extracted from one tanaidacean parasite and the two cocoons of F. cf. abyssicola by using a NucleoSpin Tissue XS Kit (Macherey–Nagel, Germany; tanaidacean parasite) or DNeasy Blood & Tissue Kits (Qiagen, Germany; cocoons). Primers 28S_1F (Álvarez-Presas et al. Reference Álvarez-Presas, Baguñà and Riutort2008) and Fe28R (newly designed; GTTTGGTTCATCCCACAGC) were used for PCR amplification (the portion containing expansion segments D1–D5 was amplified; cf. Gillespie et al. Reference Gillespie, Johnston, Cannone and Gutell2006), and 28S_1F, Fe28R, 300F, 300R (Lockyer et al. Reference Lockyer, Olson and Littlewood2003), and 28S_b5F (Kakui and Tsuyuki Reference Kakui and Tsuyuki2024) for cycle sequencing (primer 300R was not used in sequencing the cocoons). PCR amplification conditions with KOD FX Neo (Toyobo, Japan) were 94°C for 2 min; 45 cycles of 98°C for 10 s, 60°C for 30 s, and 68°C for 1 min; and 68°C for 2 min (tanaidacean parasite); and with KOD One PCR Master Mix (Toyobo) were 94°C for 2 min; 35 cycles of 94°C for 40 s, 52°C for 75 s, and 72°C for 1 min; and 72°C for 7 min (cocoons). PCR products from the tanaidacean parasite were separated on a 2% agarose gel, excised with a micro spatula, and purified with a MagExtractor PCR & Gel Clean Up Kit (Toyobo) before cycle sequencing. All nucleotide sequences were determined with a BigDye Terminator Kit ver. 3.1 and a SeqStudio Genetic Analyzer (Thermo Fisher Scientific, USA; tanaidacean parasite) by KK or at FASMAC (Kanagawa, Japan; cocoons). Fragments were concatenated by using MEGA7 (Kumar et al. Reference Kumar, Stecher and Tamura2016). The sequences we determined were deposited in the International Nucleotide Sequence Database Collaboration (INSDC) participating databases through the DNA Data Bank of Japan (DDBJ), under accession numbers LC844707 (tanaidacean parasite), and LC847139 and LC847140 (cocoons).
The 28S dataset for phylogenetic analysis included the three sequences we determined, and seven fecampiidan sequences and two outgroup sequences from the INSD (Álvarez-Presas et al. Reference Álvarez-Presas, Baguñà and Riutort2008; Gordeev et al. Reference Gordeev, Biserova, Zhukova and Ekimova2022; Hookabe et al. Reference Hookabe, Jimi, Ogawa, Tsuchiya and Sluys2023; Laumer and Giribet Reference Laumer and Giribet2014, Reference Laumer and Giribet2017; Lockyer et al. Reference Lockyer, Olson and Littlewood2003). The 28S sequences from Urastoma cyprinae (AJ313230, AY157165; Lockyer et al. Reference Lockyer, Olson and Littlewood2003; Noren and Jondelius Reference Noren and Jondelius2002) were excluded because they could be aligned with the other sequences only in a short region. The method for alignment was as described in Kakui (Reference Kakui2024a); the aligned dataset contained 868 positions (Supplementary Files S1 and S2). Methods for selecting the optimal substitution model (GTR+F+I+R2), the maximum likelihood (ML) analysis, estimation of clade support (analyses of 1000 pseudoreplicates for both Shimodaira–Hasegawa-like approximate likelihood ratio tests [SH-aLRT] and ultrafast bootstraps [UFBoot]), and drawing the tree were as described by Shimada et al. (Reference Shimada, Kakui and Fujita2023). Kimura (Reference Kimura1980) 2-parameter (K2P) distances among the aligned sequences were calculated with MEGA7.
Results and discussion
Two fecampiidans were found in the body cavity of a single host tanaidacean (BL 1.75 mm, BW 0.35 mm) (Figure 1a–d). The host was identified as a preparatory female of Pseudotanais sp. in Pseudotanaidae (cf. Jakiel et al. Reference Jakiel, Palero and Błażewicz2019; Kakui et al. Reference Kakui, Hayakawa and Katakura2017). Both parasites were cylindrical. The larger one (BL 1.10 mm, BW 0.20 mm) was light yellow, with both ends slightly deeper in color. The smaller one (BL 0.75 mm, BW 0.15 mm) was yellow. Both were completely faded in ethanol and strongly shrunken, forming a single white mass. Since the part of the mass we used for DNA extraction was from the host pleon, the 28S sequence we determined was likely from the larger individual.
Figure 1. Fecampiida flatworms parasitic in a female of the tanaidacean Pseudotanais sp. (a–d) Parasites (arrowheads) in the host, fresh (a, b) and ethanol-fixed (c, d) specimens, dorsal (a, c) and ventrolateral (b, d) views; the border between two parasites was not distinguishable after ethanol fixation. (e) Maximum-likelihood tree for Fecampiida reconstructed from 28S sequences (868 positions); numbers near nodes are Shimodaira–Hasegawa-like approximate likelihood ratio test (SH-aLRT) / ultrafast bootstrap (UFBoot) values as percentages; the scale at the bottom indicates branch length in substitutions per site.
In the 28S-based ML tree (Figure 1e), the parasite lay in a moderately well supported (SH-aLRT/UFBoot = 94.5%/84%) Fecampiidae clade. Relationships among the taxa within the clade were unclear due to lack of high nodal support. The parasite was the sister taxon to Kronborgia isopodicola, though with low support (84.5%/77%). The parasite 28S sequence was 25.0%, 32.6%, and 35.5% divergent (K2P distance) from Fecampia cf. abyssicola, Kronborgia cf. amphipodicola, and K. isopodicola sequences, respectively. Although our analysis lacked urastomid sequences, and the anatomy and cocoon shape of our parasites were unknown, based on gross morphology (i.e., the cylindrical body typical of fecampiids) and the phylogenetic position in our tree, we concluded that the parasites belong in Fecampiidae.
With 73 valid species (WoRMS 2024), Pseudotanais tanaidaceans are highly diverse and abundant in the macrobenthos, have been reported from all oceans, and show a broad depth range from several meters to 6050 m (Błażewicz et al. Reference Błażewicz, Jakiel and Bird2021; Hansen Reference Hansen1913). They are probably epifaunal or burrowers in shallow sediment (Błażewicz et al. Reference Błażewicz, Jakiel and Bird2021) or inhabit a self-woven tube in sediment (cf. Bird and Holdich Reference Bird and Holdich1989). Their high abundance and broad distribution suggest Pseudotanais as a likely candidate host group for parasites, but only nematodes had been reported to date as parasites in this genus (Błażewicz et al. Reference Błażewicz, Stępień, Jakiel, Palero, Saeedi and Brandt2020). The fecampiids in the host tanaidacean were strongly deformed and had completely lost their color in ethanol (Figure 1c, d); the lack of previous records of fecampiidans from Tanaidacea may likely have been due to simple oversight. Parasites in or on small, inconspicuous, burrowing or tube-dwelling crustaceans have been poorly investigated. As a case in point, despite their high prevalence in hosts commonly found in such an easily accessible environment as a river estuary, trematodes were first reported in a burrowing isopod (Cyathura muromiensis) only in 2024 (Shiraki and Kakui Reference Shiraki and Kakui2024). Targeted examination of such understudied groups will likely detect additional unexpected host species for fecampiidans.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X25000057.
Acknowledgements
Captain Akihisa Tsuji and the crew of RV Yokosuka; Kazuhiro Chiba and the Shinkai 6500 operation team; Hisanori Iwamoto from Nippon Marine Enterprises; Yoshihiro Fujiwara from the JAMSTEC; Dive-6K#1782 crew Takuma Onishi and Taeko Kimura; Captain Yoichi Maekawa and the crew of TRV Seisui-maru; and researchers aboard supported our collecting efforts. Taeko Kimura provided the opportunity for SS and NH to join TRV Seisui-maru cruise. Magdalena Błażewicz and Benny K. K. Chan helped in the literature survey. Matthew H. Dick reviewed the manuscript and edited our English. We are grateful to all. This study was supported by the Cooperative Research Program of the Atmosphere and Ocean Research Institute, The University of Tokyo (RV Yokosuka/DSV Shinkai 6500, Cruise YK24-09S: ‘Metapopulation structure of seamount benthos: testing the hypothesis of the Kuroshio countercurrent source off the coast of Kumano’, directed by NH).
Financial support
This study was supported by Grant-in-Aids for JSPS Fellows (JP23KJ0063 and JP23KJ2222) from the Japan Society for the Promotion of Science (JSPS).
Competing interest
The authors declare none.
